The failure to understand the pathogenesis of CF lung disease reflects, in part, our ignorance of the normal physiology of airway surface liquids (ASL). Project I proposes experiments designed to distinguish between two competing theories that purport to describe the normal physiology of ASL and measure the transport technologies interfaced with experiments using novel cell culture preparations. Specific Aim 1 sill utilize large and small airway cultures and confocal microscopy to study the physiology and ASL at the microscopic level. Key questions will focus on delineating whether there are distinct mucus and periciliary liquid layers with ASL, and whether water (PCL) moves axially toward the mouth up airways surfaces or not. Specific Aim 2 focuses on characterizing and interrelation functions of small with large airway epithelia. A comprehensive analysis of expression of genes relevant to salt secretion and absorption will be performed to test for potential regional and intra-regional patterns of absorption (proximal airways) versus secretion (glands, ?distal bronchioles). Planar and novel biofiber preparations populated with epithelia derived from microdissected bronchioles and large airways will be characterized with respect to routes of ion permeation, water permeability (L/p), volume absorption (J/v), and surface liquid ion composition. Selected parameters from the cultured preparations will be compared to data derived from freshly excised preparations. Finally, Specific Aim 3 will measure the effects of mutations in CFTR on these functions. Routes of ion permeation, L/p, and J/v will be measured in culture preparations from CF large and, if possible, small airways. A primary goal will be to measure the two parameters that discriminate between the competing theories: (1) ASL composition; and (2) existence of/ rates os isotonic J/v and its relationship to effective mucus clearance. Thus, this project should develop novel information integrating local and intra-regional ASL metabolism in health and develop key information regarding CF-specific defects in these functions that promote infection.